Blood serves as a chemical transport and communication system for the body. It contains a number of structurally and functionally diverse cellular components. The major components are red cells, platelets and white blood cells, or leukocytes. These components are sub-divided, and granulocytes comprise the major fraction of the leukocytes. Granulocytes are a vital component, providing body defenses against infection. One of the characteristic responses of the body to infection is an elevation in white blood cell count. Although infections are usually associated with an increased white cell count, decreases can also occur, especially if the bacterial invasion is massive or if the causative agent is a virus. Such decreases are usually due to a generalized depression of the bone marrow.
A rare, but potentially life-threatening complication of several drugs with otherwise beneficial therapeutic properties is their ability to inhibit granulocyte production by the bone marrow. In most cases, the development of the toxic side effect is unpredictable and does not become manifest until severe clinical complications are apparent. Thus, early recognition of an impairment in granulocyte formation, signalling the need for appropriate preventive measures, would greatly reduce the risk associated with the use of drugs known to be capable of interfering with granulocyte formation.
Predominant in granulocyte populations are the neutrophils or polymorphonuclear leukocytes. The neutrophils engulf foreign particles, for example bacterial cells, by phagocytosis which is facilitated by prior coating of the target by proteins known as opsonins. This initial step is followed by decomposition of the engulfed species by intracellular processes involving the combined action of degradative enzymes and of chemically reactive products of oxygen.
An important component in the generation of the reactive products of oxygen is the enzyme myeloperoxidase (MPO) which has been used as a marker for neutrophils. The action of MPO on intracellular chloride ions in the presence of metabolically generated hydrogen peroxide produces hypochlorous acid, which chemically degrades the foreign particles.
Important leukocyte disorders primarily involve changes in either the functional characteristics of the leukocyte or in the number of circulating leukocytes. One example of the former is chronic granulomatous disease in which granulocytes show a reduced capacity to generate reactive oxidant species in the so-called "respiratory burst" which normally destroys microorganisms that have been engulfed by phagocytic cells. Individuals affected with granulomatous disease usually die at an early age because of an inability to combat infection.
Granulocytes may increase in number in certain instances, usually in association with infection or a leukemic condition. Conversely, the clinical situation in which granulocyte numbers are reduced relative to normal also occurs. This condition, granulocytopenia, is the most frequent cause of drug or disease related alterations in body defenses against infection. Agranulocytosis, an extreme form of this disorder, is characterized by a severe and selective drop in the number of circulating neutrophils, predisposing the patient to serious and possibly even life-threatening infections. Any form of granulocyte deficiency has potentially serious clinical consequences, and early identification is crucial if the consequences of those deficiencies are to be averted.
A white blood cell count is one of the most commonly obtained laboratory values in clinical medicine. Total cell counts can be obtained by using automatic electronic devices available in clinical analytical laboratories. The quantitative assessment of individual blood cell types (the "differential count") is made by manual microscopic examination of stained blood smears or with an automated system such as a Coulter counter. Nevertheless, a white blood cell test requires a visit to a physician, a 7 milliliter venous blood sample and up to a three-day wait for laboratory results. In addition to inconvenience, delayed test results can severely affect the prognosis of a patient.
These drawbacks are compounded by virtue of white blood cell counts being probably the most commonly obtained laboratory values in clinical medicine. The cost of obtaining white blood cell counts is staggering, even not taking into account the cost of the time lost by those for whom this test is ordered.
The relative proportions of the different white blood cell types is of considerable importance in assessing the clinical implications of any leukocyte deficiency state (leukopenia). Thus, for example, a marked reduction in leukocyte levels can sometimes be fairly well tolerated provided that neutrophils comprise at least 15 to 20 percent of the total white cell population, compared with the normal range of 40 to 70 percent.
Neutrophil status is a very important diagnostic indicator, not only because of the crucial role of these cells in the defense against infection, but also because the average lifetime of neutrophils in the body is very much shorter than that of other types of blood cells such as red cells or platelets. As a result, decreased levels of circulating neutrophils can provide an early warning of impairment in bone marrow function which can occur in association with certain disease states or as a serious, and potentially fatal, complication of drug therapy or exposure to radiation. A state of neutrophil deficiency (neutropenia) is generally assumed to exist if the number of cells falls below 1,500 cells per microliter of blood. However, a considerable range of "normal" values exist for numbers of circulating leukocytes. Many blacks and Middle Eastern populations have neutrophil counts considerably lower than those of the average European.
In some cases, neutropenia can be anticipated and appropriate measures taken to minimize the risks. For example, patients receiving chemotherapy for the treatment of acute leukemia are prone to develop neutropenic enterocolitis which is caused by bacterial overgrowth in the intestinal tract. Although often limiting, this condition can damage the wall of the intestine and even result in perforation of the bowel.
In contrast to the above, predictable, dose-related risk of neutropenia for anticancer drugs (which reflects the preferential action of the drugs on rapidly multiplying cells), the development of agranulocytosis is virtually unpredictable. Agranulocytosis is a rare but potentially fatal toxic side effect of several commonly used therapeutic agents. The incidence seems to increase substantially over age 40 with an apparent increased risk in females.
Two different forms of the syndrome can be distinguished. The allergic type usually appears suddenly after the initial exposure to the drug, while the toxic variety develops insidiously over several weeks or months of continuous or intermittent therapy. Among the pharmacological agents most commonly implicated are certain antibiotics, notably the sulphonamides and chloramphenicol, antithyroid drugs (thiouracil and propylthiouracil), gold compounds, non-steroidal anti-inflammatory drugs and some antipsychotic agents, for example, chlorpromazine and clozapine.
The mechanism underlying this apparent drug sensitization is unknown. The issue of blood monitoring while a patient is being treated with an agent known to be capable of inducing agranulocytosis is clearly an important one to consider, and two salient points should be made. The first is that the onset of agranulocytosis need not be associated with the immediate development of a full-blown infection although, in the absence of routine monitoring of leukocyte status, signs of infection, notably fever or sore throat, are likely to be the first indication of the problem. As a general rule, antimicrobial therapy initiated after an infection has progressed to the symptomatic stage is likely to be less effective and to require more aggressive measures than preventative interventions. This is particularly so in the case of patients with deficient neutrophil or immune status. Secondly, continued use of an agranulocytosis-producing drug in the early, symptom-free phase of the disease can lead to a serious deterioration in the condition of the patient. Both of these conditions, therefore, require frequent assessment of leukocyte status in patients receiving drugs known to produce agranulocytosis.
There are two deterrents to this otherwise sensible, frequent monitoring of white blood cell count--cost and inconvenience. There is not currently available an inexpensive monitoring device or procedure which provides for the early detection of a significant decrease in granulocyte count that could warn of the onset of conditions such as agranulocytosis with a resultant increase in the acceptability and safety of therapeutic regimens employing drugs tending to produce this and other health and life threatening decreases in granulocyte count.
Increased circulating levels of neutrophils--for example, counts in excess of 10,000 cells per microliter of blood--are a common manifestation of infection. Microbes release chemotactic substances which can increase neutrophil activity. This involves both a stimulatory action at the level of the bone marrow and a mobilization from a less accessible marginal pool of neutrophils which are largely confined to the immediate vicinity of blood vessel walls. These partially sequestered leukocytes respond to chemical signals released from microorganisms or damaged tissues and accumulate rapidly at the site of microbial attack or injury. A typical example of the latter is the infiltration of neutrophils which is a characteristic feature of the inflammatory response.
The stimulatory action of microbial toxins on bone marrow can lead to large elevations (up to tenfold) in the number of circulating neutrophils. In some instances, such marked responses may be accompanied by the appearance of immature neutrophil precursor cells. This is referred to as the leukemoid reaction because of the similarity to a situation that exists in leukemia.
Myeloperoxidase (MPO) has been employed as a marker for granulocytes in a variety of clinical and experimental settings. This enzyme is an iron containing protein that catalyzes a reaction between hydrogen peroxide and chloride ion which produces hypochlorous acid. This reaction is important in the characteristic respiratory burst that serves to destroy bacteria following their phagocytosis by granulocytes. The absence of detectable MPO activity in other cellular elements of the blood provides a way of quantifying the severity of tissue inflammation, which is reflected in the extent of granulocyte infiltration.
Measurement of MPO activity in whole blood has also been used in the diagnosis of patients with acute leukemia and to monitor bone marrow regeneration in such patients following treatment with drugs or radiation.
Estimates of MPO activity also form the basis of automated analysis of blood smears.
The activity of MPO can readily be determined using oxidation-sensitive dyes which undergo a measurable color change during the course of a chemical reaction. The nature of the color change depends on the chemical characteristics of the particular dye that is used.
One procedure of the character just described is disclosed in Russian document No. SU 1180-001-A dated Jul. 1, 1983 (IRKUT MEDICAL INSTITUTE).
Another procedure for making a white blood cell count, also designed for laboratory settings, is disclosed in U.S. Pat. No. 3,741,875 issued Jun. 26, 1973 to Ansley et al. for PROCESS AND APPARATUS FOR OBTAINING A DIFFERENTIAL WHITE BLOOD CELL COUNT. In the Ansley et al. process, blood cells in the specimen being analyzed are killed; and the catalytic enzymes in the cells are immobilized. The cells are stained, and a photometric counter is employed to count the dyed cells.
Like others designed for laboratory settings, those procedures disclosed in the just-cited documents employ steps requiring trained personnel and specialized equipment and materials such as incubators, optical counters, zytochemical substitutes, chromogenic precipitating coupling reagents, etc.
In an outgrowth of work on the detection and estimation of glucose (see U.S. Pat. No. 2,848,308 issued Aug. 19, 1958 for COMPOSITION OF MATTER), Free et al. proposed a test for detecting leukocytes by peroxidative activity. In this test, disclosed in U.S. Pat. No. 3,087,794 issued Apr. 30, 1963 for CHEMICAL TEST FOR DIFFERENTIATING LEUKOCYTES FROM ERYTHROCYTES, contact is effected between urine which may contain both leukocytes and erythrocytes and a diagnostic composition which contains a peroxide and an indicator compound. If the sample contains leukocytes, the peroxidative enzyme will be present; and the following reactions will occur:
peroxidase (enzyme)+H.sub.2 O.sub.2 (substrate).fwdarw.intermediate compound PA1 intermediate compound+AH.sub.2 (hydrogen donor which will change color upon oxidation, reduced colorless form).fwdarw.peroxidase+H.sub.2 O+A (oxidized, colored form) PA1 patients with suppressed immune systems or with fevers and related symptoms; PA1 acute situations in emergency rooms or at home; and PA1 users of prescription drugs which are known to induce abnormal white blood cell levels (antibiotics, anti-inflammatories, antithyroids and antipsychotics).
A semiquantitative result can be obtained by measuring the time required for the color change to occur or by observing the change in intensity over a specified period of time.
The Free et al. process as applied to leukocyte analysis has very important drawbacks. One is that the test can't be applied to blood because of hemoglobin interference. Erythrocytes and leukocytes (red cells and white cells) both show peroxidative activity. The major difference is that leukocyte peroxidase is active at low concentrations of H.sub.2 O.sub.2, while the peroxidative activity of erythrocytes is due to the peroxidase-like activity of the hemoglobin, which requires a much higher concentration of H.sub.2 O.sub.2 to become operative. Therefore, the color reaction at a low concentration of H.sub.2 O.sub.2 is due to the presence of leukocytes and the color reaction at a high concentration of H.sub.2 O.sub.2 is due to the presence of red cells (especially since myeloperoxidase tends to be inhibited by high concentrations of H.sub.2 O.sub.2).
A second major drawback of the Free et al. procedure is that no provision is made for releasing material with oxidative reactivity from the cells potentially present in the specimen before the determination of leukocyte activity is made. This deficiency is significant because the material in question is typically present inside the cell instead of on the cell surface, the marker enzyme myeloperoxidase for granulocytes being typical in this respect. Absent anything for releasing such materials, therefore, a false white blood cell count will almost certainly be obtained.
Another significant drawback of the Free et al. process as applied to the making of white blood cell counts is that no provision is made for eliminating interference from other blood components. Perhaps the most abundant source of this contamination is the enzyme catalase found in erythrocytes (red blood cells). Catalase can promote the same peroxide decomposition reactions as white blood cell markers such as myeloperoxidase. Consequently, absent a step to keep this from happening, a change in the color of an oxidation sensitive indicator as used by Free et al. may not reflect with any degree of accuracy the activity of leukocyte in a specimen being analyzed.
In short there is a continuing, important and unfilled need for a simple, inexpensive, easy-to-use, reliable procedure for making white blood cell counts and for a diagnostic kit implementing such a procedure which does not require skilled personnel or special facilities or equipment and can be used in home and other nonclinical settings with a minimum of instruction. This would, as one example, materially facilitate the treatment of infections as the efficacy of a selected course of treatment as a reflection of a change in granulocyte count could be much more easily ascertained. A procedure of the character in question would also significantly increase the ability to track the effect of those pharmacological agents which have the potential of producing a harmful change in white blood cell count.
There is a comparable need for methods and kits as characterized in the preceding paragraph which are sufficiently accurate and efficacious to be useful in diagnostic and monitoring protocols by more highly trained personnel in field, clinical and other settings.